柑橘采后青霉病原中寄生病毒的筛选与鉴定

Screening and identification of parasitic viruses in Penicillium pathogen of postharvest citrus

【目的】从采后霉烂柑橘上分离青霉病原菌,并筛选和鉴定病原中寄生的青霉病毒。【方法】以柑橘园腐烂的柑橘为材料,挑取分生孢子在PDA培养基上进行划线分离,根据总基因组电泳图谱筛选具有基因组额外核酸片段的病毒菌株,并使用RT-PCR扩增额外片段测序鉴定相应病毒。【结果】根据菌落形态初步分组,分离到两类菌落特征差异明显的青霉菌(共25株),其中具灰绿色菌落的青霉16株,具暗黄绿色菌落的青霉9株。扩增两类青霉的代表菌株(DY10和DY18)的ITS并测序,鉴定结果显示,DY10和DY18分别为意大利青霉(Penicillium italicum)和指状青霉(P.digitatum)。青霉菌株总基因组电泳筛选发现,DY9、DY11、DY12、DY17菌株中存在约6000 bp的基因组额外片段,推测可能为指状青霉病毒1 (PdV1)同种不同病毒株的基因组。根据PdV1基因组设计特异性引物,RT-PCR扩增出约500 bp的条带,测序序列与PdV1基因组同源序列的一致性高达99.7%,将所分离的病毒鉴定为PdV1同种的不同病毒株。【结论】本研究从腐烂柑橘上分离鉴定意大利青霉16株和指状青霉9株,并从指状青霉菌株中筛选和鉴定出4株与维多利亚病毒属(Victorivirus) PdV1同种的不同病毒株,为柑橘绿霉病的绿色防控提供潜在的生防病毒资源。

【Objective】The study intends to isolate the Penicillium strains from postharvest mold citrus,and identify the parasitic mycovirus in Penicillium pathogen.【Method】Using decaying fruits in citrus orchard as materials,conidia were selected and separated by streaking on PDA medium.Virus strains with additional nucleic acid fragments of the genome were screened according to total genome electrophoresis map,and the corresponding viruses were identified by RT-PCR amplification of additional fragments.【Result】According to the colony morphology,two types of Penicillium(25 strains in total)with distinct colony characteristics were isolated,including 16 strains of Penicillium with grey-green colonies and 9 strains of Penicillium with dark yellow-green colonies.Two representative strains of P.italicum and P.digitatum(DY10 and DY18)were amplified by ITS and the PCR product was sequenced.The identification results showed that they were P.italicum and P.digitatum,respectively.The total genome of Penicillium strains possessed an additional element with a size of about 6000 bp.It was specu-lated that it might be homologous to the reported PdV1 virus genome.Using Pd V1 genome as a reference sequence,a primer pair was de-signed to amplify c DNA from the dsRNA by RT-PCR.The PCR product was a specific fragment with a size of about 500 bp spanned CP to RdRp encoding genes partial sequences,which was sequenced to obtain a sequence of 444 bp by Sanger sequencing.The BLASTx alignment showed that the c DNA sequence derived from the viral dsRNA was 99.7%identity with the homology region of the P.digitatum virus 1(KU257669.1,KU933932.1).【Conclusion】Four strains of P.digitatum virus were identified as different virus strains of the same species as PdV1 belonging to the genus Victorivirus.The research provides more biocontrol virus resources for the study of the interaction mechanism between mycovirus and the host fungi,and the green prevention and control of the citrus green mold.